Staphylococcus aureus, a major cause of nosocomial and medical device infections, exists either as planktonic cells or as adherent, complex communities known as biofilms. S. aureus biofilm infections evade cellular host defenses to a greater extent than planktonic infections; this contributes to difficultly in treating biofilm infections. Cytokines released from activated host cells have opposing effects on bacterial survival, both recruiting and activating host cells to eliminate bacteria, but also directly promoting bacterial growth. Progress in understanding the bacterial mechanisms that increase growth in response to cytokines is hampered by the loss of cytokine-responsiveness after in vitro passage. Since biofilm growth resembles in vivo growth and is often implicated in recalcitrant infections, we hypothesize that increased bacterial growth in response to mammalian cytokines is dependent upon the biofilm phenotype. If this hypothesis is true, the increased bacterial growth in response to the cytokines secreted by inflammatory cells may help explain why biofilm infections are more difficult to treat. Our proposal has three aims. First, we will determine whether S. aureus responsiveness to the major proinflammatory cytokines secreted by activated macrophages is due to growth as a biofilm (Specific Aim #1). This will be accomplished by preparing biofilm and planktonic cultures of S. aureus and using fluorescent dyes and plate counting to measure bacterial growth in response to cytokines. Second, we will identify the S. aureus receptors that bind cytokines (Specific Aim #2). Cytokine binding will be assessed by flow cytometry; co-precipitation, proteomics, and transposon mutagenesis will be used to identify cytokine receptor genes. Transposon mutagenesis or allelic replacement will be used to prepare cytokine receptor mutants. Third, we will study the regulation of the cytokine receptors, and determine the contribution of biofilm cytokine binding to the increased evasion of host cellular defenses by bacteria (Specific Aim #3). RT-PCR will be used to examine the expression of cytokine receptor genes during biofilm development, and co-cultures of wild-type and cytokine receptor mutants with macrophages will demonstrate the role of cytokine receptors in host cell evasion. Determining the conditions necessary to study S. aureus responsiveness to cytokines will allow for the elucidation of the molecular mechanisms involved, and may lead to new molecular targets for the treatment of S. aureus biofilm infections. [unreadable] [unreadable] [unreadable]